Covalent phosphorylation of proteins plays a central role in the control of many cellular processes by hormones such as insulin and epinephrine. Part of this regulation involves the enzymes responsible for dephosphorylating proteins, the phosphoprotein phosphatases. This project addresses one of the main classes of protein phosphatase found in cells, what will be termed "type 1" phosphatase (one characteristic of such enzymes is that their catalytic subunit is inhibited by the heat stable phosphatase inhibitor proteins, inhibitor-1 and inhibitor-2). Knowledge of the structure and regulation of these enzymes is incomplete. A primary objective will be to establish the number and structures of the native forms of this class of enzyme. Methods to be applied will include (1) conventional protein purification, (2) immunological methods, and (3) recombinant DNA techniques. Purified high molecular weight forms of type 1 phosphatase will be characterized and compared with known enzymes of simpler structure, namely the isolated catalytic subunit, C1, and the ATP-mg-dependent phosphatase. The functional roles of non-catalytic subunits will be investigated, particularly in relation to control of enzyme activity. Special attention will be paid to phosphorylation of phosphatases since there is good reason to believe that phosphorylation of the inhibitor-2 component of the ATP-Mg-dependent phosphatase is related to its activation. Possible controls mediated by small molecules will be evaluated. Models for the short-term intracellular control of phosphatase activity will employ mouse diaphragms exposed to insulin and/or epinephrine. Immunological methods will be applied to analyze the phosphorylation of inhibitor-2, and other phosphatase components, as well as phosphatase activity. Success in this investigation can have widespread importance since this class of phosphatase is likely to be involved in the control of many cellular processes. Any mechanistic link to the action of hormones, especially insulin, will be of particular interest in understanding how insulin functions normally. Impaired phosphatase, however, could be a determining factor in some types of diabetes.